Anti-cancer compounds from invasive salvinias and methods of treating cancer

ABSTRACT

Compositions and methods related to anti-cancer terpenoid compounds isolated from plants in the Salviniaceae family, pharmaceutical compositions comprising the same, and methods of treating cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/US2014/016724, filed Feb. 17, 2014,which claims the benefit of priority of U.S. Provisional PatentApplication No. 61/769,506, filed on Feb. 26, 2013, The entire contentsof each of the above-referenced disclosures are specificationincorporated herein by reference without disclaimer.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant number2008-38928-19308 awarded by the U.S. Department of Agriculture. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention generally relates to compositions derived from plants andpharmaceutical uses thereof. In particular, the invention relates to theuse of bioactive composition isolated from plants in the Salviniaceaefamily.

B. Description of Related Art

Giant salvinia (Salvinia molesta D. S. Mitchell) is one of the mostwidespread and environmentally, economically and socially destructiveinvasive plant species in the world. The salvinia also provides habitatfor snails that are intermediate hosts for Schistosoma sp. which causesparasitic disease Schistosomiasis, the second most socioeconomicallydevastating parasitic disease after malaria. To date, control measures,particularly biological and chemical methods of this invasive speciesare very expensive and have failed to achieve their purpose and maycause environmental backlashes. Positive control by harvesting andutilizing giant salvinia has never been developed.

Therefore, new beneficial uses and positive control methods for theseplants are needed.

SUMMARY OF THE INVENTION

In some embodiments, the compositions and methods disclosed hereinrelate to compounds isolated from plants in the Salviniaceae family,compositions comprising the same, and methods of using the same.

In one aspect, there are provided compounds of the formula:

wherein R₁ is H, OH, OCH₃, or ═O; R₂ is H, OH, OCH₃, or ═O; R₃ is H, OH,or OCH₃; and R₄ is H, or OH.

In one aspect, there are provided compounds of the formula:

wherein R₁ and R₂ are, independently, CHO, CH₃OH, or COOH.

In embodiments, the compounds are further defined as:

In one aspect, there are provided compounds of the formula:

wherein R₁ is H, OH, OCH₃, or ═O; R₃ is H, OH, or OCH₃; R₄ is H or OH;and R₂ is:

In embodiments, the compounds are further defined as:

In one aspect, there are provided compounds of the formula:

wherein R1 is one of:

R₂ is H or OH; and R₃, R₄, and R₅ are each, independently, H, OH, orOMe.

In embodiments, the compounds are further defined as:

In embodiments, the compounds are further defined as:

wherein R₁ is α-H or OH; and R₂ is β-H or OH.

In one aspect, there are provided compounds of the formula:

wherein R is β-D-glucopyranosyl or H.

In one aspect, there are provided compounds of the formula:

In one aspect, there are provided compounds of the formula:

In some aspects, the compound is isolated from a plant in theSalviniaceae family. In some embodiments, the compound is isolated froma plant in the Salvinia genus. In some embodiments, the compound isisolated from S. auriculata, S. biloba, S. cucullata, S. cyathiformis,S. hastate, S. herzogii, S. minima, S. molesta, S. natans, S.nymphellula, S. oblongifolia, S. radula, S. rotundifolia, or S. sprucei.In some embodiments, the compound is isolated from a plant in the Azollagenus.

In some aspects, there are provided pharmaceutical compositionscomprising one or more of the above compounds and an excipient. In otheraspects there are provided methods of treating cancer in patients inneed thereof, comprising administering to such patients one or more ofthe above compounds in an amount sufficient to treat the cancer.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dogs,cat, mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses. In someembodiments, the patient is a human.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease. In some embodiments, treating cancer isfurther defined as reducing the size of a tumor or inhibiting growth ofa tumor.

The compounds may be administered by any acceptable route. In someembodiments, the compounds are administered orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intrapleurally,intraprostaticaly, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,orally, parenterally, rectally, subconjunctival, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incremes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof.

The cancer may be any cancer. In some embodiments, the cancer ismelanoma, cervical cancer, breast cancer, ovarian cancer, prostatecancer, testicular cancer, urothelial carcinoma, bladder cancer,non-small cell lung cancer, small cell lung cancer, sarcoma, colorectaladenocarcinoma, gastrointestinal stromal tumors, gastroesophagealcarcinoma, colorectal cancer, pancreatic cancer, kidney cancer,hepatocellular cancer, malignant mesothelioma, leukemia, lymphoma,myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma,neuroblastoma, plasma cell neoplasms, Wilm's tumor, or hepatocellularcarcinoma. In some embodiments, the cancer is pancreatic cancer,leukemia, lung cancer, breast cancer, or prostate cancer.

The compositions may be administered one or more times. In someembodiments, the compositions are administered 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 times or more.

The methods of the present invention may be used in combination withadditional cancer therapy. In some embodiments, the distinct cancertherapy comprises surgery, radiotherapy, chemotherapy, toxin therapy,immunotherapy, cryotherapy or gene therapy. In some embodiments, thecancer is a chemotherapy-resistant or radio-resistant cancer.

“Effective amount” or “therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease. In someembodiments, the subject is administered at least about 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 mg/kg (or any range derivable therein).

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

In some aspects, there are provided methods of treating liver or cardiacdisease in a patient in need thereof, comprising administering to thepatient one or more of the above compounds in an amount sufficient totreat the liver or cardiac disease.

The term “about” or “approximately” are defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. Compositions and methods“consisting essentially of” any of the ingredients or steps disclosedlimits the scope of the claim to the specified materials or steps whichdo not materially affect the basic and novel characteristic of theclaimed invention.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Bioassay guided isolation procedure of Salvinia molesta.

FIG. 2. Structures of the compounds isolated from Salvinia molesta.

FIGS. 3A-D. FIG. 3A Effect of salviniol (6) on the proliferation ofhuman exocrine pancreatic cancer cells (BxPC-3). FIG. 3B Effect ofmontbretol (8) on the proliferation of human exocrine pancreatic cancercells (BxPC-3). FIG. 3C Effect of salviniol (6) on the proliferation ofhuman exocrine pancreatic cancer cells (PANC-1). FIG. 3D Effect ofmontbretol (8) on the proliferation of human exocrine pancreatic cancercells (PANC-1).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The ethanol extract of giant salvinia and other salvinia species showspromising activity to control human tumor growth with less toxicity tonormal cells in vitro. The inventors have isolated over 50 compoundsfrom the plants including six new compounds and identified a class ofbioactive compounds. These compounds showed in vitro bioactivity againstnon-small cell lung cancer (A549 (human lung adenocarcinoma epithelialcell line)), prostate cancer (PC-3 (human prostate adenocarcinomaepithelial cell line)), pancreatic cancer (non-endocrine PANC-1 (humanexocrine pancreatic carcinoma) and exocrine BxPC-3 (human exocrinepancreatic carcinoma)), breast cancer (MDA-MB-231 (human breastadenocarcinoma epithelial cell line)), and leukemia (HL-60 (humanpromyelocytic leukemia cell line)) cells.

The bioactive compounds, particularly the new compound salviniol (6) (arare abietane diterpene with a new ferruginol-menthol coupled skeleton)isolated from giant salvinia provide lead compounds in the drugdevelopment for cancer and inflammation treatment. Pharmaceuticalproduction of bioactive agents from salvinia will provide the best wayto “control” the most noxious invasive species.

A. SALVINIACEAE

Salviniaceae is a family of heterosporous ferns in the orderSalviniales. The Salviniaceae contain the two genera Azolla andSalvinia.

Azolla, also known as mosquito fern, duckweed fern, fairy moss, waterfern, is a genus of seven species of aquatic ferns. They are extremelyreduced in form and specialized, looking nothing like conventional fernsbut more resembling duckweed or some mosses.

Salvinia Ség. is a genus of floating ferns having 10-14 species in theworld, particularly in the tropics. Salvinia molesta D. S. Mitchell,known as giant salvinia, water fern, or kariba weed, is native toBrazil. Since 1939, it has invaded lake and river systems in warmclimates in the world (Room, 1990). At present, giant salvinia is one ofthe most widespread and environmentally, economically and sociallydestructive invasive plant species (Schooler, 2011). In addition, giantsalvinia provides habitat for snails that are intermediate hosts forSchistosoma sp. which parasitize the human intestinal and urinarytracts. The parasitic disease Schistosomiasis is also known as snailfever, bilharzia, or bilharziosis, is the second most socioeconomicallydevastating parasitic disease after malaria.

Giant salvinia is able to double in number and biomass in less thanthree days under optimal condition and forms dense mats over stillwaters (Barrett, 1989). The plant can regenerate vegetatively even aftersevere damage or drying for days (Room, 1986; Finlayson, 1984). Theexplosive growth of S. molesta does not only affect adversely thenatural ecological system of the infested region but also causeconsiderable economic damage and sanitation problems. Dense mats of S.molesta reduce dissolved oxygen levels and block all sunlight frompenetrating the infested water body. Thus, macrophytes and microscopicalgae that form the base of the food chain may die off (Room, 1990;USDA: Giant Salvinia-Pest Alert). The creatures that feed on these maydie, too, and so on up the food chain. This pest threatens cultivatedaquatic crops, and it can clog irrigation and drinking water lines andfoul hydroelectric plants. Salvinia infested waters cannot be used forboating or other recreational purposes (USDA: Giant Salvinia-PestAlert).

Since 1980, the tiny salvinia weevil (Cyrtobagous salviniae,Curculionidae) has been introduced into most regions where giantsalvinia has invaded (Julien, 2009). The weevil is a strict specialistwith adults feeding on salvinia buds while the plant is highlysusceptible to the insects and thus the weevil has successfullycontrolled salvinia for years in some regions. But recently it was foundthat the biological control is incomplete and fitful associated withstochastic flooding events and thus is unpredictable (Schooler, 2011).Other attempts to control and eradicate S. molesta through chemical andmechanical means have failed to achieve their purpose and may causeenvironmental backlashes being caused by the introduction of chemicalsor bioagents into the environment (Abbasi, 1986).

Researchers had analysed the chemical composition of giant salvinia toevaluate its suitability as a source of forage for ruminants but thehigh content of crude ash, lignin and tannins restrict the use(Moozhiyil, 1986). Recently, it was found that salvinia is able toaccumulate certain metals makes it potentially useful for wastemanagement and effluent treatment (Choudhary, 2008). Positive control byharvesting and utilizing giant salvinia has not been developed, and themedical or pharmaceutical potential of giant salvinia remainsunexplored. Most studies on cancer drug discovery have been prioritizedon non-common plant species, and no species of the genus Salvinia orfamily Salvinaceae has been previously reported to have anti-tumoractivity.

Previous phytochemical investigations reported six phenolics, oneiridiod, and one terpenoid in the Salvinia genus and some compoundsshowed antioxidant activity (Choudhary, 2008; Srilaxmi, 2010;Chantiratikul, 2009; Narasimjulua, 2010).

B. THERAPEUTIC COMPOUNDS

A crude ethanol extract of S. molesta showed moderate cytotoxic activityagainst human non-small cell lung cell (A549), human prostate cancercell (PC-3), and human pancreatic cancer cell (PANC-1) but not toxic tonormal human lung fibroblasts cells (MRC-5). A bioactivity-guidedfractionation of ethanol extracts of giant salvinia led to the isolationof 50 compounds including 17 abietane diterpenes (6-22), nine phenolics(2, 4, 5, 27-30, 43, and 44), four apocavotenoids (36-39), fiveunsaturated hydroxyl fatty acids (24-26, 45, and 46), two acyclicsesquiterpenoids (1 and 23), two monoterpenes (3 and 40), two jasmonates(31 and 32), two steroids (34 and 35), two coumarins (41 and 42), andfive triterpenes (33, and 47-50). All abietane diterpenes were isolatedfrom Salvinia for the first time. Their structures were elucidated byspectroscopic data interpretation. Among the 50 compounds, six are newcompounds (1-6). Salviniol [7-(menth-1-en-4-ol)-ferruginol] (6) is arare abietane diterpene with a ferruginol-menthol coupled skeleton.

Cytotoxicity of 50 compounds against human cancer cell lines wasexamined in vitro. Among all compounds, 16 abietane diterpenes (6-17 and19-22) demonstrated activities against human non-small cell lung cancer(A549), prostate cancer (PC-3), exocrine pancreatic cancer (PANC-1 andBxPC-3), breast cancer (MDA-MB-231), and leukemia (HL-60) cells andnormal human lung fibroblasts MRC-5. The bioactivities of compounds 6,11, 15, 16, 17, and 22 were reported for the first time. Thebioactivities of compounds 7, 8, 12, 20, and 21 against human exocrinepancreatic cancer have never been reported before. It is also the firstreport of the selective activities of compounds 7 and 8 to humannon-small cell lung cancer (A549) but less toxic normal human lungfibroblasts MRC-5. The cytotoxicity of compounds 12 and 21 against humannon-small cell lung cancer (A549), prostate cancer (PC-3), and leukemia(HL-60) are reported for the first time. See Table 1.

Based on the structure-activity relationship analysis of bioactivecompounds, the abietane diterpenes with group “OH”, “OCH₃” or “═O” atC-12 have potent cytotoxicity against human tumors, and abietanediterpenes also have one of these groups at C-6 will enhance thebioactivity. 6,7-seco abietane derivatives possess cytotoxicity, butshow less activity than the regular abietanes according to the bioassayresults.

TABLE 1 Compound No. Name Structure Characteristics and Activity 6Salviniol (7-(menth-1-en-4- ol)-ferruginol)

A rare abietane diterpene with a new ferruginol- menthol coupledskeleton 1. Activity against human exocrine pancreatic tumor PANC-1(GI₅₀ = 60.44 ± 16.17 μM) and BxPC-3 (GI₅₀ = 87.28 ± 24.51 μM) 2.Activity against human non-small cell lung tumor A549 (GI₅₀ = 62.91 ±9.63 μM) 3. Activity against human prostate tumor PC-3 (GI₅₀ = 54.19 ±5.90 μM) 4. Activity against human leukemia HL-60 (GI₅₀ = 48.05 ± 10.86μM) 5. Activity against human breast tumor MDA-MB-231 (GI₅₀ = 75.0 ±1.37 μM) 7 14-Deoxycoleon U

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 22.59± 4.18 μM) and BxPC-3 (GI₅₀ = 37.28 ± 7.44 μM) 2. The compound is lesstoxic to normal human lung fibroblasts MRC-5 (GI₅₀ = 24.75 ± 4.73 μM)than to human non-small cell lung tumor A549 (GI₅₀ = 9.10 ± 2.47 μM) 3.This compound is selective and has no activity to breast tumor(MDA-MB-231, GI₅₀ > 100 μM) 8 Montbretol

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 23.08± 9.16 μM) and BxPC-3 (GI₅₀ = 25.0 ± 13.19 μM) 2. The compound is lesstoxic to normal human lung fibroblasts MRC-5 (GI₅₀ = 20.01 ± 5.14 μM)than to human non-small cell lung tumor A549 (GI₅₀ = 11.07 ± 3.16 μM) 95,6-Dehydrosugiol

1. Activity against human non-small cell lung tumor A549 (GI₅₀ = 84.52 ±18.73 μM) 10 7-Methoxyrosmanol

1. Activity against human non-small cell lung tumor A549 (GI₅₀ = 86.50 ±9.54 μM) 11 Montbretyl 12- methyl ether

1. Activity against human non-small cell lung tumor A549 (GI₅₀ = 21.21 ±6.21 μM) 12 11-Hydroxysugiol

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 43.14± 8.32 μM) and BxPC-3 (GI₅₀ = 45.01 ± 8.33 μM) 2. Activity against humannon-small cell lung tumor A549 (GI₅₀ = 35.04 ± 6.20 μM) 3. Activityagainst human prostate tumor PC-3 (GI₅₀ = 41.03 ± 7.90 μM) 4. Activityagainst human leukemia HL-60 (GI₅₀ = 35.26 ± 0.63 μM) 15 7-Hydroxyferruginol

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 78.24± 13.46 μM) and BxPC-3 (GI₅₀ = 74.73 ± 11.09 μM) 2. Activity againsthuman non-small cell lung tumor A549 (GI₅₀ = 75.11 ± 7.04 μM) 3.Activity against human prostate tumor PC-3 (GI₅₀ = 85.61 ± 5.30 μM) 4.Activity against human leukemia HL-60 (GI₅₀ = 75.19 ± 1.02 μM) 16 6,7-Dehydroferruginol

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 46.71± 10.98 μM) and BxPC-3 (GI₅₀ = 37.50 ± 4.01 μM) 2. Activity againsthuman non-small cell lung tumor A549 (GI₅₀ = 53.79 ± 9.65 μM) 3.Activity against human prostate tumor PC-3 (GI₅₀ = 58.12 ± 3.28 μM) 4.Activity against human leukemia HL-60 (GI₅₀ = 66.52 ± 0.04 μM) 1712-Hydroxy simonellite

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 37.53± 5.72 μM) and BxPC-3 (GI₅₀ = 36.42 ± 8.08 μM) 2. Activity against humannon-small cell lung tumor A549 (GI₅₀ = 42.22 ± 3.89 μM) 3. Activityagainst human prostate tumor PC-3 (GI₅₀ = 43.60 ± 11.88 μM) 4. Activityagainst human leukemia HL-60 (GI₅₀ = 50.01 ± 15.23 μM) 20 Royleanone

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 52.55± 18.84 μM) and BxPC-3 (GI₅₀ = 51.04 ± 10.44 μM) 21 6,7-Dehydroroyleanone

1. Activity against human exocrine pancreatic tumor PANC-1 (GI₅₀ = 51.34± 10.04 μM) and BxPC-3 (GI₅₀ = 41.74 ± 2.92 μM) 2. Activity againsthuman non-small cell lung tumor A549 (GI₅₀ = 47.96 ± 7.75 μM) 3.Activity against human prostate tumor PC-3 (GI₅₀ = 46.32 ± 6.60 μM) 4.Activity against human leukemia HL-60 (GI₅₀ = 30.03 ± 8.30 μM) 2212-Hydroxy-6,7- secoabieta-8,11,13- triene-6,7-dial

1. Activity against human non-small cell lung tumor A549 (GI₅₀ = 93.75 ±21.39 μM) 4 Salviniside I

Novel benzofuran glucose conjugates with unique 10-membered macrodiolidestructures 5 Salviniside II

Novel benzofuran glucose conjugates with unique 10-membered macrodiolidestructures

The bioactive diterpanes isolated from salvinias have extremely lowconcentrations (ppm levels) which makes the isolation and elucidation ofthese compounds from salvinias difficult. For example, from in total11.3 kg of dried giant salvinia matters (about 250 kg in fresh weight),inventors obtained only 2.0 mg of Salvinol (compound 6). Theconcentration of salviniol (6) in giant salvinia is about 0.178 ppm indry weight. Other active compounds have similar concentrations.Therefore, such very minor compounds would be ignored by mostphytochemists and would not be obtained by routine phytochemicalanalysis.

Because the extremely low contents of bioactive compounds in salviniatissues, the salvinia extracts demonstrated moderate activities againsthuman tumor cells. Such low activities made salvinias ignored in generalplant screening for anti-tumor agents.

C. DEFINITIONS

When used in the context of a chemical group, “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl,—Br or —I; “amino” means —NH₂; “hydroxyamino” means —NHOH; “nitro” means—NO₂; imino means ═NH; “cyano” means —CN; “isocyanate” means —N═C═O;“azido” means —N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂or a deprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH;“thio” means ═S; “sulfonyl” means —S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond; and “≡” means triple bond. The symbol “----”represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, thestructure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond. The symbol “

”, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the conformation (e.g., either R or S) orthe geometry is undefined (e.g., either E or Z).

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the groups and classes below, the following parenthetical subscriptsfurther define the group/class as follows: “(Cn)” defines the exactnumber (n) of carbon atoms in the group/class. “(C≦n)” defines themaximum number (n) of carbon atoms that can be in the group/class, withthe minimum number as small as possible for the group in question, e.g.,it is understood that the minimum number of carbon atoms in the group“alkenyl_((C≦8))” or the class “alkene_((C≦8))” is two. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. The term does not preclude carbon-heteroatommultiple bonds, for example a carbon oxygen double bond or a carbonnitrogen double bond. Moreover, it does not preclude a carbon-carbondouble bond that may occur as part of keto-enol tautomerism orimine/enamine tautomerism.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).When the term “aliphatic” is used without the “substituted” modifieronly carbon and hydrogen atoms are present. When the term is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched, cyclo, cyclic or acyclic structure,and no atoms other than carbon and hydrogen. Thus, as used hereincycloalkyl is a subset of alkyl. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “alkanediyl” when usedwithout the “substituted” modifier refers to a divalent saturatedaliphatic group, with one or two saturated carbon atom(s) as thepoint(s) of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “alkylidene”when used without the “substituted” modifier refers to the divalentgroup ═CRR′ in which R and R′ are independently hydrogen, alkyl, or Rand R′ are taken together to represent an alkanediyl having at least twocarbon atoms. Non-limiting examples of alkylidene groups include: ═CH₂,═CH(CH₂CH₃), and ═C(CH₃)₂. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.The following groups are non-limiting examples of substituted alkylgroups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃,—CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂,and —CH₂CH₂Cl. The term “haloalkyl” is a subset of substituted alkyl, inwhich one or more hydrogen atoms has been substituted with a halo groupand no other atoms aside from carbon, hydrogen and halogen are present.The group, —CH₂Cl is a non-limiting examples of a haloalkyl. An “alkane”refers to the compound H—R, wherein R is alkyl. The term “fluoroalkyl”is a subset of substituted alkyl, in which one or more hydrogen has beensubstituted with a fluoro group and no other atoms aside from carbon,hydrogen and fluorine are present. The groups, —CH₂F, —CF₃, and —CH₂CF₃are non-limiting examples of fluoroalkyl groups. An “alkane” refers tothe compound H—R, wherein R is alkyl.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and no atoms other than carbon and hydrogen.Non-limiting examples of alkenyl groups include: —CH═CH₂ (vinyl),—CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and—CH═CH—C₆H₅. The term “alkenediyl” when used without the “substituted”modifier refers to a divalent unsaturated aliphatic group, with twocarbon atoms as points of attachment, a linear or branched, cyclo,cyclic or acyclic structure, at least one nonaromatic carbon-carbondouble bond, no carbon-carbon triple bonds, and no atoms other thancarbon and hydrogen. The groups, —CH═CH—, —CH═C(CH₃)CH₂—, —CH═CHCH₂—,and

are non-limiting examples of alkenediyl groups. When these terms areused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups. An “alkene” refersto the compound H—R, wherein R is alkenyl.

The term “alkynyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one carbon-carbon triple bond, and no atoms otherthan carbon and hydrogen. As used herein, the term alkynyl does notpreclude the presence of one or more non-aromatic carbon-carbon doublebonds. The groups, —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃, are non-limitingexamples of alkynyl groups. When alkynyl is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. An“alkyne” refers to the compound H—R, wherein R is alkynyl.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl group (carbon number limitation permitting) attached tothe first aromatic ring or any additional aromatic ring present.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, and themonovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic group,with two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl group (carbon number limitation permitting) attached to the firstaromatic ring or any additional aromatic ring present. If more than onering is present, the rings may be fused or unfused. Non-limitingexamples of arenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. An “arene” refers to thecompound H—R, wherein R is aryl.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group-alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the“substituted” modifier one or more hydrogen atom from the alkanediyland/or the aryl has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. Non-limiting examples ofsubstituted aralkyls are: (3-chlorophenyl)-methyl, and2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein,the term does not preclude the presence of one or more alkyl, aryl,and/or aralkyl groups (carbon number limitation permitting) attached tothe aromatic ring or aromatic ring system. If more than one ring ispresent, the rings may be fused or unfused. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl,pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term“heteroarenediyl” when used without the “substituted” modifier refers toan divalent aromatic group, with two aromatic carbon atoms, two aromaticnitrogen atoms, or one aromatic carbon atom and one aromatic nitrogenatom as the two points of attachment, said atoms forming part of one ormore aromatic ring structure(s) wherein at least one of the ring atomsis nitrogen, oxygen or sulfur, and wherein the divalent group consistsof no atoms other than carbon, hydrogen, aromatic nitrogen, aromaticoxygen and aromatic sulfur. As used herein, the term does not precludethe presence of one or more alkyl, aryl, and/or aralkyl groups (carbonnumber limitation permitting) attached to the aromatic ring or aromaticring system. If more than one ring is present, the rings may be fused orunfused. Non-limiting examples of heteroarenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. As used herein, the term does notpreclude the presence of one or more alkyl groups (carbon numberlimitation permitting) attached to the ring or ring system. If more thanone ring is present, the rings may be fused or unfused. Non-limitingexamples of heterocycloalkyl groups include aziridinyl, azetidinyl,pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, andpyranyl. When the term “heterocycloalkyl” used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl orheteroaryl, as those terms are defined above. The groups, —CHO, —C(O)CH₃(acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂,—C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅, —C(O) (imidazolyl) arenon-limiting examples of acyl groups. A “thioacyl” is defined in ananalogous manner, except that the oxygen atom of the group —C(O)R hasbeen replaced with a sulfur atom, —C(S)R. When either of these terms areused with the “substituted” modifier one or more hydrogen atom(including the hydrogen atom directly attached the carbonyl orthiocarbonyl group) has been independently replaced by —OH, —F, —Cl,—Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The groups,—C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃,—C(O)NH₂ (carbamoyl), and —CON(CH₃)₂, are non-limiting examples ofsubstituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy), —OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —OCH(CH₂)₂,—O-cyclopentyl, and —O-cyclohexyl. The terms “alkenyloxy”, “alkynyloxy”,“aryloxy”, “aralkoxy”, “heteroaryloxy”, and “acyloxy”, when used withoutthe “substituted” modifier, refers to groups, defined as —OR, in which Ris alkenyl, alkynyl, aryl, aralkyl, heteroaryl, and acyl, respectively.The term “alkoxydiyl” refers to the divalent group —O-alkanediyl-,—O-alkanediyl-O—, or -alkanediyl-O-alkanediyl-. The term “alkylthio” and“acylthio” when used without the “substituted” modifier refers to thegroup —SR, in which R is an alkyl and acyl, respectively. When any ofthese terms is used with the “substituted” modifier one or more hydrogenatom has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂,—C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The term “alcohol” corresponds to analkane, as defined above, wherein at least one of the hydrogen atoms hasbeen replaced with a hydroxy group.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃ and —NHCH₂CH₃. The term “dialkylamino” when used without the“substituted” modifier refers to the group —NRR′, in which R and R′ canbe the same or different alkyl groups, or R and R′ can be taken togetherto represent an alkanediyl. Non-limiting examples of dialkylamino groupsinclude: —N(CH₃)₂, —N(CH₃)(CH₂CH₃), and N-pyrrolidinyl. The terms“alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, and “alkylsulfonylamino” when usedwithout the “substituted” modifier, refers to groups, defined as —NHR,in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, andalkylsulfonyl, respectively. A non-limiting example of an arylaminogroup is —NHC₆H₅. The term “amido” (acylamino), when used without the“substituted” modifier, refers to the group —NHR, in which R is acyl, asthat term is defined above. A non-limiting example of an amido group is—NHC(O)CH₃. The term “alkylimino” when used without the “substituted”modifier refers to the divalent group ═NR, in which R is an alkyl, asthat term is defined above. The term “alkylaminodiyl” refers to thedivalent group —NH-alkanediyl-, —NH-alkanediyl-NH—, or-alkanediyl-NH-alkanediyl-. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ are non-limiting examples ofsubstituted amido groups.

The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the“substituted” modifier refers to the groups —S(O)₂R and —S(O)R,respectively, in which R is an alkyl, as that term is defined above. Theterms “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”,“aralkylsulfonyl”, and “heteroarylsulfonyl”, are defined in an analogousmanner. When any of these terms is used with the “substituted” modifierone or more hydrogen atom has been independently replaced by —OH, —F,—Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2 n, where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diasteromers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≦15%, morepreferably ≦10%, even more preferably ≦5%, or most preferably ≦1% ofanother stereoisomer(s).

“Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” means that amount which, whenadministered to a subject or patient for treating a disease, issufficient to effect such treatment for the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease. In some embodiments, treatment of apatient afflicted with one of the pathological conditions describedherein comprises administering to such a patient an amount of compounddescribed herein which is therapeutically effective in controlling thecondition or in prolonging the survivability of the patient beyond thatexpected in the absence of such treatment. As used herein, the term“inhibition” of the condition also refers to slowing, interrupting,arresting or stopping the condition and does not necessarily indicate atotal elimination of the condition. It is believed that prolonging thesurvivability of a patient, beyond being a significant advantageouseffect in and of itself, also indicates that the condition isbeneficially controlled to some extent.

Other abbreviations used herein are as follows: ¹H-NMR is proton nuclearmagnetic resonance, AcOH is acetic acid, Ar is argon, CH₃CN isacetonitrile, CHN analysis is carbon/hydrogen/nitrogen elementalanalysis, CHNCl analysis is carbon/hydrogen/nitrogen/chlorine elementalanalysis, CHNS analysis is carbon/hydrogen/nitrogen/sulfur elementalanalysis, DI water is deionized water, DIC is diisopropyl carbodiimide,DMA is N,N-dimethylacetamide, DMAP is 4-(N,N-dimethylamino)pyridine, DMFis N,N-dimethylformamide, EDCl is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EtOAc isethyl acetate, EtOH is ethanol, FAB MS is fast atom bombardment massspectroscopy, g is gram(s), h is hour, HOBT is 1-hydroxybenzotriazolehydrate, HPLC is high performance liquid chromatography, IBCF isisobutylchloroformate, KSCN is potassium thiocyanate, L is liter, LiOHis lithium hydroxide, MEM is methoxyethoxymethyl, MEMCl ismethoxyethoxymethyl chloride, MeOH is methanol, mg is milligram, MgSO₄is magnesium sulfate, mL is milliliter, mL is milliliter, min is minute,MS is mass spectroscopy, MTBE is methyl tert-butyl ether, N₂ isnitrogen, NaHCO₃ is sodium bicarbonate, NaOH is sodium hydroxide, Na₂SO₄is sodium sulfate, NMM is N-methylmorpholine, NMP is N-methylpyrrolidinone, NMR is nuclear magnetic resonance, P₂O₅ is phosphorouspentoxide, PTSA is para-toluenesulfonic acid, RPHPLC is reverse phasehigh performance liquid chromatography, RT is room temperature, TFA istrifluoroacetic acid, THF is tetrahydrofuran, TMS is trimethylsilyl, μgis microgram, and Δ is heating the reaction mixture.

D. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

For administration to a mammal in need of such treatment, the compoundsin a therapeutically effective amount are ordinarily combined with oneor more excipients appropriate to the indicated route of administration.The compounds may be admixed with lactose, sucrose, starch powder,cellulose esters of alkanoic acids, cellulose alkyl esters, talc,stearic acid, magnesium stearate, magnesium oxide, sodium and calciumsalts of phosphoric and sulfuric acids, gelatin, acacia, sodiumalginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and tabletedor encapsulated for convenient administration. Alternatively, thecompounds may be dissolved in water, polyethylene glycol, propyleneglycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil,benzyl alcohol, sodium chloride, and/or various buffers. Otherexcipients and modes of administration are well and widely known in thepharmaceutical art.

The pharmaceutical compositions useful in the present invention may besubjected to conventional pharmaceutical operations such assterilization and/or may contain conventional pharmaceutical carriersand excipients such as preservatives, stabilizers, wetting agents,emulsifiers, buffers, etc.

The compounds of the present disclosure may be administered by a varietyof methods, e.g., orally or by injection (e.g. subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, the active compounds may be coated in a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes.

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in apatient. For example, the efficacy of a compound can be evaluated in ananimal model system that may be predictive of efficacy in treating thedisease in humans, such as the model systems shown in the examples anddrawings.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1,000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day,500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 90,00 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. Forexample, regarding treatment of diabetic patients, the unit dosage maybe an amount that reduces blood glucose by at least 40% as compared toan untreated subject. In another embodiment, the unit dosage is anamount that reduces blood glucose to a level that is ±10% of the bloodglucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1μg/kg/body weight, about 5 μg/kg/body weight, about 10 μg/kg/bodyweight, about 50 μg/kg/body weight, about 100 μg/kg/body weight, about200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/bodyweight, about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/bodyweight, about 200 mg/kg/body weight, about 350 mg/kg/body weight, about500 mg/kg/body weight, to about 1,000 mg/kg/body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5μg/kg/body weight to about 500 mg/kg/body weight, etc., can beadministered, based on the numbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.1% of a compoundof the present disclosure. In other embodiments, the compound of thepresent disclosure may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may be takenorally and that the timing of which is or is not dependent upon foodintake. Thus, for example, the agent can be taken every morning and/orevery evening, regardless of when the subject has eaten or will eat.

E. COMBINATION THERAPY

In addition to being used as a monotherapy, the compounds of the presentinvention may also find use in combination therapies. Effectivecombination therapy may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, administered at the same time,wherein one composition includes a compound of this invention, and theother includes the second agent(s). Alternatively, the therapy mayprecede or follow the other agent treatment by intervals ranging fromminutes to months.

Non-limiting examples of such combination therapy include combination ofone or more compounds of the invention with another anti-inflammatoryagent, a chemotherapeutic agent, radiation therapy, an antidepressant,an antipsychotic agent, an anticonvulsant, a mood stabilizer, ananti-infective agent, an antihypertensive agent, a cholesterol-loweringagent or other modulator of blood lipids, an agent for promoting weightloss, an antithrombotic agent, an agent for treating or preventingcardiovascular events such as myocardial infarction or stroke, anantidiabetic agent, an agent for reducing transplant rejection orgraft-versus-host disease, an anti-arthritic agent, an analgesic agent,an anti-asthmatic agent or other treatment for respiratory diseases, oran agent for treatment or prevention of skin disorders. Compounds of theinvention may be combined with agents designed to improve a patient'simmune response to cancer, including (but not limited to) cancervaccines.

F. EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the applicants to function well inthe practice of the invention. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials and Methods

General Experimental Procedures

NMR experiments were performed on a JEOL ECS-400 and a Bruker Avance 700NMR instrument. NMR data were reported as δ (ppm) values and referencedto the solvent used. HRESIMS were acquired on an electrospray instrument(MDS Sciex Pulsar Qstar, Ontario, Canada). Octadecyl-functionalizedsilica gel, silica gel, Sephadex LH-20, and TLC plates were purchasedfrom Aldrich Chemical Co. HPLC analysis was performed on a HewlettPackard Series 1100 with a HP 1100 diode array detector using a SB-C18ODS column (250×4.6 mm, 5 μM, Agilent). Preparative HPLC was performedwith an Acuflow Series III pump connected with an Acutect 500 UV/VISdetector using an Econosil ODS column (250×22 mm, 10 μM, Alltech).D-Glucose was purchased from Aldrich.

Collection, Extraction, and Isolation

The whole plants of S. molesta were collected. Air-dried plant material(11.26 kg) was ground to a coarse powder and extracted 48 hrs with 95%EtOH (100 L×2) at room temperature. The combined EtOH extracts wereconcentrated to give a residue (480 g) under reduced pressure. Theresidue was then suspended in MeOH/H₂O (1,500 mL, 1:1, v/v) andextracted successively with Hexane, EtOA cand n-BuOH. The Hexane andEtOAc extracts exhibited strong cytotoxic activity against A549 cellline with GI₅₀ value of 48.61 μg/mL and 54.83 μg/mL, respectively, andthus was subjected to further purification by cytotoxicity directedfractionation against A549 cell line.

The hexane extract (53 g) was applied on a column of silica gel (1,100g) eluting with the mixture of CH₂Cl₂/MeOH (50:1, v/v, 5 L). Fractionswere monitored by TLC and similar fractions combined to afford 5fractions, of which fraction 24B and 24C showed the best activity. Afterbeing combined, fractions 24B and 24C were further fractionated by ODScolumn chromatography (1,000 g) eluting with MeOH/H₂O (15:85, 30:70,50:50, and 75:25, v/v, each 3 L), and 1 L of 100% MeOH. According to theHPLC profiles, the eluents were combined to give five subfractions: 25A,25B, 25C, 25D, and 25E. Fraction 25D afforded eight compounds 7 (2.9mg), 8 (3.4 mg), 10 (12.9 mg), 11 (2.1 mg), 12 (2.0 mg), 13 (5.7 mg), 43(3.1 mg), and 44 (5.9 mg), respectively, by preparative HPLC (MeOH/H₂O,80:20, v/v). Fraction 25E afforded eight compounds 6 (2.0 mg), 14 (9.6mg), 15 (2.0 mg), 16 (2.8 mg), 17 (13.8 mg), 19 (2.4 mg), 20 (2.0 mg),and 21 (2.0 mg), respectively, by preparative HPLC (MeOH/H₂O, 82:18,v/v). Compound 18 (1.9 mg) was purified from 25C by preparative HPLC(MeOH/H₂O, 99:1, v/v). Compounds 34 (1.0 mg), 35 (1.0 mg), and 22 (10.5mg) were obtained from 25B by preparative HPLC (MeOH/H₂O, 95:5, v/v),respectively. Compounds 33 (17.0 mg), 47 (3.0), and a sub-fraction 25A1were obtained from 25A by recrystallization in hexane/acetone.Sub-fraction 25A1 was then further isolated by analytical HPLC with 100%MeOH to give compounds 48 (1.2 mg), 49 (1.5 mg), and 50 (1.0 mg).

The EtOAc-soluble extract (83.9 g) was applied to a column of DiaionHP-20 eluting with H₂O, MeOH—H₂O (30:70, 45:55, 60:40, and 80:20), andMeOH to give five fractions: F30 (30% methanol elution, 25 g), F45 (45%methanol elution, 13 g), F60 (60% methanol elution, 13.6 g), F80 (80%methanol elution, 7.8 g), and F100 (methanol elution, 7.8 g). Thesefractions were evaluated for cytotoxicity against A549 cell line, ofthese, fraction F45 and F60 showed the most potent inhibition of cancercell proliferation. Faction F60 was separated by silica gel columnchromatography (500 g) eluted with a CH₂Cl₂/MeOH gradient (7:1, 3:2,2:3, and 1:99, v/v, each 3 L) to afford compounds 45 (5.0 mg) and 46 (23mg). The rest 47 fractions were combined into fractions FI6-FVI6. Afterbeing combined, FI6 and FII6 were separated by preparative HPLC to givecompounds 9 (8.0 mg), 24 (1.3 mg), 25 (1.2 mg) and 26 (1.8 mg) elutedwith CH₃CN/0.5% HOAc in H₂O (54:46, v/v). Fraction FIV6 was applied to aODS column eluting with MeOH/H₂O (1:1, v/v) to afford six sub-fractions:FV6a-FV6f. Compounds 23 (3.0 mg), 27 (22 mg), 30 (2.3 mg), and 32 (1.0mg) were purified by preparative HPLC from fraction FV6c (MeOH/H₂O,54:46, v/v). HPLC isolation of FV6f eluted with MeOH/H₂O (35:65, v/v)gave compounds 4 (600 mg) and 5 (10.9 mg). After being combined,fractions FV6 and FVI6 were further fractionated by HPLC eluting withMeOH/H₂O (30:70) to give compounds 1 (2.2 mg) and 28 (1.0 mg). FactionF45 was separated by silica gel column chromatography (500 g) elutedwith a gradient CH₂Cl₂/MeOH (10:1, 5:1, 3:2, and 2:3, v/v, each 3 L) and1 L of MeOH, to give five fractions FI4-FV4. Compounds 29 (1.2 mg) and31 (1.1 mg) were purified by preparative HPLC from fraction FIV4 bypreparative HPLC (MeOH/H₂O, 30:70, v/v). Faction F80 (7.8 g) wasseparated by silica gel column chromatography eluted with a CH₂Cl₂/MeOHgradient (15:1, 10:1, 5:1, 2:1 and 0:1, v/v, each 1.5 L) to afford 6fractions F8a-F8f. F8b was subjected to an ODS column, eluted with agradient of MeOH/H₂O (from 40:60 to 90:10), to give three subfractions,F8b1-F8b3. F8b1 was subjected to a silica gel column, eluted with agradient of hexane/acetone (from 10:1 to 2:1), to yield compounds 2 (6mg), 41 (5.2 mg), and 42 (1.9 mg). F8b2 was subjected to a silica gelcolumn, eluted with a gradient of hexane/EtOAc (from 4:1 to 0:1), togive three fractions F8b2a-F8b2c. Compounds 36 (4.2 mg), 39 (7 mg), and40 (9 mg) were purified by preparative HPLC from fraction F8b2b bypreparative HPLC (MeOH/H₂O, 70:30, v/v). F8c was subjected to a silicagel column, eluted with a gradient of hexane/acetone (from 10:1 to 2:1),to yield compounds 3 (6 mg), 37 (5.2 mg), and 38 (9 mg).

Hydrolysis and Determination of Absolute Configuration of Sugar

Compound 4 (10.0 mg) and 5 (5.0 mg), respectively, was refluxed with 2mL of 5% H₂SO4 at 80° C. for 3 h (Toyota, 2002). After cooling, thereaction mixture was diluted with water, and then partitioned threetimes with EtOAc (each 5 mL). The remaining aqueous solution wasevaporated to dryness with nitrogen gas stream. Compound 1 (1.0 mg) wasincubated in 400 μL 1 M HCl in dioxane/water (1:1, v/v) at 80° C. for 3h. The solution was evaporated to dryness by blowing nitrogen gas. Theresidue was suspended in 200 μL water and partitioned three times withEtOAc (400 μL×3). The remaining aqueous solution was evaporated todryness with nitrogen gas stream.

The absolute configuration of sugar in each residue was determined by amethod described by Tanaka et al with minor modifications (Tanaka,2007). Briefly, the sample was dissolved in 200 μL pyridine containingL-cysteine methyl ester hydrochloride (equivalent to the theoreticalweight of sugar in the sample) and incubated at 60° C. After 1 hrincubation, 200 μL o-torylisothiocyanate (equivalent to the theoreticalweight of sugar in the sample) pyridine solution was added into themixture and incubated at 60° C. for one additional hour. The reactionmixture was directly analyzed by HPLC (Agilent 1100 HPLC system,Poroshell 120 EC-C18, 4.6×50 mm, 2.7 μm, 0.5 mL/min, 250 nm, 22%acetonitrile in 50 mM H₃PO₄, then washing column with 90% acetonitrilein 50 mM H₃PO₄). By HPLC analysis, the retention time of the productfrom the standard sugar was found at 15.7 min for D-glucose. Bycomparison of the retention time with the standard and co-HPLC, theabsolute configuration of sugar in the hydrolysis was identified.

Cytotoxicity Assays

Human exocrine pancreatic cancer (PANC-1 and BxPC-3), non-small celllung cancer (A549), prostate cancer (PC-3), breast cancer (MDA-MB-231),and leukemia (HL-60) were obtained from The University of Texas M. D.Anderson Cancer Center, Houston, Tex., and Normal human lung fibroblastscells (MRC-5) was purchased from the American Type Culture Collection(ATCC). PC-3, A549 and HL-60 cells were maintained in RPMI 1640 mediumsupplemented with 10% (v/v) fetal bovine serum and antibiotics (1,000units/mL penicillin-streptomycin solution, Hyclone). PANC-1 and MRC-5was cultured in complete growth medium according to the ATCC protocols.Cells were grown at 37° C. and 5% CO2 in humidified air.

Cytotoxicity of total extracts of S. molesta was tested against PANC-1,A549, PC-3, and MRC-5. Cytotoxicity of the fractions was determinedusing A549 cells, a human non-small lung cancer cell line. Fractionswere tested at 25, 50, and 100 μg/mL. The isolated pure compounds weretested in each of the cultured human tumor and normal cell lines atvarious concentrations between 1 and 100 μM with three replications.Human tumor and normal cells were inoculated into 96 well microtiterplates in 90 μL at plating densities ranging from 5,000 to 40,000cells/well (measured by Z2 Coulter Counter of Beckman) depending on thedoubling time of individual cell lines. Cell growth and viability wasmeasured by WST-8 (Water Soluble Tetrazolium) assays following astandard protocol (Cell Counting Kit-8, Dojindo Molecular, Maryland).The WST-8 assay measured the relative activity of mitochondrialreductase enzymes in viable cells. A control WST reading (T₀) beforetreatment was measured, then cells were continuously exposed to isolatesfor 48 h. After WST added and incubated for 2 h, the amount of formazanwas measured by using a microplate spectrophotometer (μQuantspectrophotometer, Bio-Tek Instruments, Inc., Vermont) at 450 nm. T_(C)or T_(D) represents the readings of the untreated control or with testedcompounds. Percent cell-growth (G) and percent cell-growth inhibition(GI) was calculated as: G=(T_(D)−T₀/T_(C)−T₀)×100;GI=[1−(T_(D)−T₀/T_(C)−T₀)]×100 for which T_(D)≧T₀; andG=(T_(D)−T₀/T₀)×100; GI=[1−(T_(D)−T₀/T₀)]×100 for which T_(D)<T₀. Todetermine the inhibitory potency of active compounds (GI₅₀, 50%cell-growth inhibition), tests were extended to additionalconcentrations (varying from 0.01 to 100 μM). Cancer drug doxorubicinwas used as the positive controls in the cell-based assays.

Cell Proliferation Assays

CCK-8 (Dojindo Molecular, Maryland) was used to measure theproliferation response of salviniol (6) and montbretol (8) against humanexocrine pancreatic cancer cell lines (PANC-1 and BxPC-3), according tothe manufacturer's instructions. Cell were grown in growth medium plus10% FBS and 5% antibiotics in 96-well plates and then were treated withvarious concentrations of test compounds and incubated for 12, 24, 48,and 72 h, respectively. The end of the incubation period, 10% CCK-8solution was added to the wells. After 2 h incubation, the absorbance at450 nm was measured. A calibration curve was prepared using theabsorbance observed known numbers of viable cells.

Example 2

The EtOH extract of S. molesta was found to exhibit moderate selectivecytotoxicity against several human cancer cell lines (A549: GI₅₀ 84.34μg/mL; PC-3: GI₅₀ 100 μg/mL; PANC-1: GI₅₀ 69.92 μg/mL), but no activityfor normal lung cell line (MRC-5: GI₅₀>100 μg/mL). This prompted theinventors to phytochemically examine the chemical constituentsresponsible for the cytotoxic activity using bioactivity-guidedfractionation (FIG. 1). This paper reports the isolation, structuralelucidation, and cytotoxic activity of six new compounds,10(S)-hydroxy-2,6,10-trimethyl-2E,6E,11-dodecatrienoic acid10-O-β-D-glucopyranoside (1), salvinin A (2), salvinin B (3),salviniside I (4), salviniside II (5), and salviniol(7-(menth-1-en-4-ol)-ferruginol) (6) together with 16 abietanediterpenes: 14-deoxycoleon U (7) (Kusumoto, 2009; Fraga, 2005),montbretol (8) (Ayhan, 1992), 5,6-dehydrosugiol (9) (Xu, 2011),7-methoxyrosmanol (10) (Guerrero, 2006), montbretyl 12-methyl ether (11)(Ulubelen, 1992), 11-hydroxysugiol (12) (Xiang, 2002), sugiol (13)(Benjamin, 2003), ferruginol (14) (Tezuka, 1998), 7-hydroxyferruginol(15) (Kuo, 2002), 6,7-dehydroferruginol (16) (Kuo, 1998), 12-hydroxysimonellite (17) (Elisa, 1969), simonellite (18) (Elisa, 1969),1-oxomiltirone (19) (Sairafianpour, 2001), royleanone (20) (Tezuka,1998), 6,7-dehydroroyleanone (21) (Kusumoto, 2009),12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (22) (Fang, 1986) and28 other known compounds:10(S)-hydroxy-2,6,10-trimethyl-2E,6E,11-dodecatrienoic acid (23)(Miyazawa, 1996), 6-hydroxy-7E,9Z-pentadecadienoic acid (24) (Tamura,2010), 11-hydroxy-7Z,9E,13E-hexadecatrienoic acid (25) (Xiang, 2008),13-hydroxy-9Z,11E,15E-octadecatrienoic acid (26) (Bang, 2002), 4-hydroxymethyl benzoate (27), 3,4-dihydroxy benzoic acid (28), 4-hydroxy benzoicacid (29), p-hydroxybenzaldehyde (30) (Avupati, 2012), cucurbic acid(31) (Fujita, 1996), cucurbinoyl-isoleucine (32) (Hans-Dieter, 1995),hop-22(29)-ene (33) (Mahato, 1994), β-sitosterol (34), β-stigmasterol(35), (3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one (36) (DellaGreca,2004), annuionone D (37) (Zhao, 2011), dehydrovomifoliol (38) (Tan,2012), (+)-3-hydroxy-β-ionone (39) (DellaGreca, 2004), pubinernoid A(40) (Huang, 2006), 3-methoxycoumarin (41) (Mustafa, 1963),4-methyl-7-ethyl-coumarin (42) (Brubaker, 1986), asiaticin (43)(Siddiuui, 2007), 2-(benzoyloxy)-1,1-dimethyl-ethyl (44) (Fielding,2002), 13-hydroxy-tetradecanoic acid methyl ester (45), decanoic acid(46) (Yu, 1999), 3β-acetoxy-20(29)-hopene (47), 3β-acetoxy-hopane (48),3β-acetoxy-29-hydroxy-hopane (49), 3β-acetoxy-16-hopene (50) (FIG. 2).The identities of these known compounds were determined by analyzingtheir spectroscopic data and confirmed by comparing their values withthose in the literature.

Compound 1 was isolated as a pale, amorphous powder. The molecularformula C₂₁H₃₃O₈ was deduced from the [M-H]⁺ peak at m/z 413.2174 (calcdfor 413.2175) in the HR-ESI-MS. The ¹³C NMR spectrum of 1 displayed 21carbon signals, of which 15 were assigned to the aglycone, including 4quaternary carbons [one carbonyl (δ 171.12), two olefinic quaternarycarbon (δ 134.29 and 127.93), and one oxyquaternary carbons (δ 80.50)],and the remaining 6 to the sugar moiety (Table 2).

TABLE 2 ¹³C and ¹H NMR data of compound 1 (in MeOH-d₄)^(a) 1 Positionδ_(C) (ppm) δ_(H) (ppm) 1 171.12 2 127.93 3 142.19 6.68 (1H, t, J = 6.67Hz) 4 26.87 2.26 (1H, dd, J = 7.33 and 7.99 Hz) 5 37.93 2.09 (1H, t, J =7.33 Hz) 6 134.29 7 125.13 5.14 (1H, t, J = 7.79 Hz) 8 22.21 2.06 (1H,m) 9 41.13 1.56 (1H, m) 10  80.50 11  143.09 5.90 (1H, dd, J = 10.99 and17.40 Hz) 12  114.14 5.18 (1H, dd, J = 17.40 and 1.37 Hz) 5.00 (1H, dd,J = 10.99 and 1.37 Hz) 13  11.14 1.77 (3H, s) 14  14.52 1.59 (3H, s) 15 21.93 1.35 (3H, s) Glc-1′ 98.18 4.31 (1H, d, J = 7.79 Hz) Glc-2′ 73.753.13 (1H, m) Glc-3′ 76.60 3.12 (1H, m) Glc-4′ 70.29 3.25 (1H, t, J =8.70 Hz) Glc-5′ 76.34 3.28 (1H, m) Glc-6′ 61.40 3.61 (1H, dd, J = 5.50,11.91 Hz) 3.78 (1H, dd, J = 2.37, 11.91 Hz) ^(a)The assignment was basedupon COSY, HSQC, and HMBC experiments.

An anomeric carbons (δ 98.18) and an anomeric protons at δ 4.31 (1H, d,J=7.79 Hz) observed in the HSQC spectrum, in addition to signals of fouroxymethine groups and an oxymethylene group at δ 73.75 (CH, C-2′), 76.60(CH, C-3′), 70.29 (CH, C-4′), 76.34 (CH, C-5′), and 61.40 (CH2, C-6′)suggested the sugar as β-glucopyranose (Zhang, 2008), which was furtherdetermined to be β-D-glucopyranose by co-HPLC analysis with authenticsugar after acid hydrolysis of compound 1 (Tanaka, 2007). In the ¹H NMRspectrum, signals for three methyls appearing as singlet peaks [δ_(H)1.35, 1.59, 1.77], four methylene, two olefinic methines [δ_(H) 6.68(1H, t, J=6.67 Hz), 5.14 (1H, t, J=7.79 Hz)], and a terminal vinyl grouphaving a small geminal coupling (J=1.37 Hz), a trans vicinal coupling(J=17.40 Hz), and a cis vicinal coupling (J=10.99 Hz) indicated thepresence of a sesquiterpene moiety. Its structural fragment wasdetermined by 2D NMR data, including COSY, HSQC, and HMBC experiments.From the COSY spectrum of compound 1, it was possible to establish theproton sequence from H-3 to H₂-5 through H₂-4; H-7 to H₂-9 through H₂-8;and H-11 to H₂-12. The methyl groups attached at C-2, C-6, and C-10 weredetermined on the basis of the key HMBC correlations from H₃-13 to C-1,C-2, and C-3; H₃-14 to C-5, C-6, and C-7; and H₃-15 to C-9, C-10, andC-11. The above HMBC correlations also confirmed the linkagesestablished by the COSY experiment. The NMR data comparison of compounds1 and 23 concluded that compound23 was the aglycone of compound 1identificated as 10(S)-hydroxy-2,6,10-trimethyl-2,6,11-dodecatrienoicacid (Miyazawa, 1996). The long-range correlation of δ_(H) 4.31 (GlcH-1) of the glucose (Glc) with δ_(C) 80.50 (C-10) of the skeleton fromHMBC correlations suggested that Glc was located at C-10 position. TheE- and E-geometry of the two double bonds at C-2 and C-6 were identifiedby the NOESY correlations between H-13/H-4 and H-5, H3-14 and H-8. Thestructure of compound 1 was formulated as10(S)-hydroxy-2,6,10-trimethyl-2E,6E,11-dodecatrienoic acid10-O-β-D-glucopyranoside.

Salvinin A (2), obtained as a pale yellow powder. The molecular formulaC₁₀H₈O₂ was established by HR-EI-MS and 1 D NMR spectrum (Table 3).

TABLE 3 ¹³C and ¹H NMR data of compound 2^(a) 2 Position δ_(C) (ppm)δ_(H) (ppm) 1 195.6 2 126.1 3 132.6 8.17, s 4 136.5 5 121.3 8.19 (1H,dd, J = 8.4, 2.4 Hz) 6 121.9 7.20, m 7 123.0 7.20, m 8 110.6 7.42 (1Hdd, J = 7.6, 2.2 Hz) 9 113.6 10 64.9 4.73, s ^(a)The assignment wasbased upon COSY, HSQC, and HMBC experiments.

The ¹H-NMR spectrum of compound 2 displayed the signals of five sp2protons and two sp3 protons. The ¹³C-NMR spectrum with DEPT experimentsdisplayed 10 carbon resonances comprising one oxygenated methylene, fivesp2 methines, and four quaternary carbons (one keto group). The ¹H-NMRspectrum (δ 8.19, dd, J=8.4 and 2.4 Hz, H-5; δ 7.20, m, H-6 and H-7; δ7.42, dd, J=7.6 and 2.2 Hz, H-8) revealed an o-disubstituted benzenering substructure. HMBC correlations of H₂-10 to C-1, C-2 and C-3,indicated that the only oxygenated methylene (C-10) was placed at C-2.Thus, compound 2 was elucidated as 2-hydroxymethyl, Indone.

Salvinin B (3), obtained as an amorphous powder. The molecular formulawas established as C₁₁H₁₆O₂ by HR-ESI-MS and 1 D NMR spectrum. The¹H-NMR spectrum (Table 4) showed three methyls at δ 1.25, 1.29, and 1.57as singlets, and one trisubstituted double bond at δ 5.75. The ¹³C-NMRspectrum (Table 4) showed 11 carbon signals, identified by a DEPTexperiment as three methyls, two methylenes, one oxymethine, one doublebond carbon, two quaternary carbons and one carbonyl carbon.

TABLE 4 ¹³C and ¹H NMR data of compound 3^(a) 3 Position δ_(C) (ppm)δ_(H) (ppm) 1 172.6 2 112.4 5.75, s 3 182.6 4 87.2 5 47.6 2.45, dt, J =12.4, 5.2 Hz; 1.41, m 6 63.9 4.07, m 7 47.9 1.97 dt, J = 12.4, 2.8 Hz;1.36, m 8 34.8 10 29.0 1.25, s 11 24.0 1.29, s 12 24.4 1.57, s ^(a)Theassignment was based upon COSY, HSQC, and HMBC experiments.

The ¹H—¹H COSY spectrum together with the HSQC data revealed a—CH₂—CH—CH₂— unit in bold (3a). HMBC correlations of H₃-12/C-3 and C-4,H-2/C-1, C-3, C-4 and C-8, H₃-11/C-1, C-8 and C-10 suggested the partialstructure 3b (FIG. 2). The two substructures were linked via C-4 andC-5, C-7 and C-8 by the HMBC correlations of H₃-11/C-7 and H₃-12/C-5,respectively. The only leftover uncertainty for the planar structure of3 was the remaining one degree of unsaturation, which required thepresence of an additional ring. The relatively upfield shifted of the¹³C NMR data at C-1 at δ 172.6 and the relatively downfield shifted ofC-6 at δ 63.9, suggesting that an ether bridge was present between C-1and C-6 to form an oxygen ring. The relative stereochemistry of compound3 was established on the basis of the ROESY experiment. The strong ROESYcorrelations of H-5β/H₃-12 and H-6β indicated that H-6, and Me-12 wereβ-oriented. The other configurations were established as the same ascompound pubinernoid A (40) by comparing their spectroscopic data(Huang, 2006). The gross structure of compound 3 was thus established as(4S*,6S*)-4-hydroxy-4,8,8-trimethyl-9-oxabicyclo[4.2.1]non-1-en-3-one.

Compound 4 was obtained as colourless powder and was assigned amolecular formula of C₂₇H₂₆O₁₃, as deduced from the [M-H]^(|) peak atm/z 557.1297 (calcd for 557.1295) in the HR-ESI-MS. The ¹³C and ¹H-NMRspectrum of compound 4 (Table 5) displayed a set of glycoside signals(δ_(C) 103.7, 77.7, 75.6, 74.5, 68.7, 65.7). The anomeric proton at δ4.39 with coupling constant J_(H1,2) value (d, J=7.8 Hz) demonstratedthe presence of a β-glucopyranosyl moiety (Narasimhulua, 2010). Themonosaccharide was further determined to be D configuration afterchemical degradation of compound 4 (Oiso, 2001; Toyota, 2002) and HPLCanalysis with authentic sugar. The ¹H-NMR spectrum of compound 4contained signals due to two oxygenated methylenes at δ 4.11 (2H, d,J=6.4 Hz, H-4′″) and 4.06, 4.21 (1H each, d, J=12.2 Hz, H-1′″), a vinylmethyl group and an olefinic proton at δ 1.70 (3H, s, H-5′″) and 5.65(1H, dd, H-3′″). Thus, compound 4 possessed a hemiterpene with atrisubstitued double bond (Ding, 1999; Kazuko, 2006). Five aromaticprotons were also found in the ¹H-NMR spectrum (Table 5), of which threesignals at δ 7.39 (1H, dd, J=1.8 and 8.2 Hz, H-2′), δ 6.83 (1H, d, J=8.2Hz, H-3′), and 7.45 (1H, d, J=1.8 Hz, H-6′) were characteristic of a1,2,4-trisubstituted phenyl moiety, the remaining two at δ 7.67 (1H, d,J=8.2 Hz, H-6) and 6.79 (1H, d, J=8.2 Hz, H-7) were assigned to a1,2,3,4-tetra substituted phenyl group (Narasimhulua, 2010).

TABLE 5 ¹H and ¹³C-NMR Data (δ) of Compounds 4 and 5^(a) 5 4 β-anomerα-anomer Position δ_(C) δ_(H) δ_(C) δ_(H) δ_(C) δ_(H)  2 158.4 158.3158.4  3 110.2 110.3 110.2  4 129.7 129.7 129.7  5 114.8 114.9 114.8  6130.0 7.67 (1H, d, J = 8.2 Hz) 130.0 7.70 (1H, d, J = 8.2 Hz) 130.0 7.70(1H, d, J = 8.2 Hz)  7 112.1 6.79 (1H, d, J = 8.2 Hz) 112.1 6.82 (1H, d,J = 8.2 Hz) 112.1 6.82 (1H, d, J = 8.2 Hz)  8 149.0 148.8 148.8  9 143.5143.5 143.5 10 168.5 168.7 168.7 11 168.0 168.1 168.1  1′ 121.6 121.6121.6  2′ 121.4 7.39 (1H, dd, J = 8.2 and 121.5 7.39 (1H, dd, J = 8.5and 121.4 7.39 (1H, dd, J = 8.5 and 1.8 Hz) 1.8 Hz) 1.8 Hz)  3′ 116.76.83 (1H, d, J = 8.2 Hz) 116.8 6.84 (1H, d, J = 8.5 Hz) 116.8 6.84 (1H,d, J = 8.5 Hz)  4′ 149.4 149.4 149.4  5′ 146.8 146.8 146.8  6′ 115.77.45 (1H, d, J = 1.8 Hz) 115.7 7.45 (1H, d, J = 1.8 Hz) 115.7 7.45 (1H,d, J = 1.8 Hz) Glc-1″ 103.7 4.39 (1H, d, J = 7.8 Hz) 98.6 4.57 (1H, d, J= 7.8 Hz) 94.4 5.14 (1H, d, J = 3.7 Hz) Glc-2″ 75.6 3.32 (1H, dd J = 8.2and 76.7 3.27 (1H, dd, J = 7.8 and 74.2 3.51(1H, dd, J = 3.7 and 8.7 Hz)9.2 Hz) 9.2 Hz) Glc-3″ 74.5 3.59 (1H, dd, J = 9.6 and 74.5 3.57 (1H, dd,J = 9.6 and 71.6 3.83 (1H, t, J = 10.1 Hz) 9.2 Hz) 9.2 Hz) Glc-4″ 77.75.27 (1H, t, J = 9.6 Hz) 77.8 5.30(1H, t, J = 9.6 Hz) 78.4 5.24 (1H, t,J = 9.6 Hz) Glc-5″ 68.7 3.77 (1H, td, J = 10.1 and 68.9 3.78 (1H, td, J= 10.5 and 64.4 4.24 (1H, td, J = 10.5 and 4.1 Hz) 4.1 Hz) 4.1 Hz)Glc-6″ 65.7 3.93 (1H, t, J = 10.5 Hz) 65.8 3.94 (1H, t, J = 11.0 Hz)66.4 3.91 (1H, t, J = 11.0 Hz) 5.02 (1H, dd, J = 10.5 and 5.04 (1H, dd,J = 10.3 and 4.97(1H, dd, J = 10.3 and 4.1 Hz) 4.1 Hz) 4.1 Hz)  1″′ 75.84.06 (1H, d, J = 12.2 Hz); 4.21 (1H, d, J = 12.2 Hz)  2″′ 135.8  3″′128.5 5.65 (1H, dd, J = 5.9 and 6.4 Hz)  4″′ 59.2 4.11 (2H, d, J = 6.4Hz)  5″′ 14.2 1.70 (3H, s) ^(a)The assignment was based upon COSY, HSQC,and HMBC experiments.

A close inspection of the ¹³C-NMR spectrum of compound 4, together withthe MS data, clearly revealed that the 1,2,3,4-tetrasubstituted phenylmoiety should be a part of a 8-carbon benzofuran skeleton (Hsieh, 2006;Kazuko & Kentaro, 2006). In additional, the ¹³C NMR spectra of compound4 displayed two ester carbonyl carbons at δ 168.0 and δ 168.5. Asobserved in the HMBC spectrum of compound 4, three key HMBC correlationamong δ_(H) 4.39 (glc H-1″) and δ_(C) 75.8 (hemiterpene C-1′″)established that the glucose was attached to the assigned position; H-6at δ_(H) 7.67 and Glc H-6″ at δ_(H) 3.93 with a carbonyl carbon at δ_(C)168.0 (C-11) established the linkage between the benzofuran unit andH-6″ of Glc. Similarly, the other ester carbonyl carbon (δ_(C) 168.5,C-10) was attached to H-4″ of Glc position by the HMBC spectrumanalysis. These long-range HMBC correlations of H-2′ to C-1′, C-6′ andC-2, and of H-6′ to C-1′, C-2′ and C-2 established the connectivity ofthe benzofuran skeleton with 1,2,4-trisubstituted phenyl moiety. The1,1-ADEQUATE spectrum also confirmed the assignment. The relativelydownfield shift of C-3 at δ 110.2 and the remaining one degree ofunsaturation established the linkage of the C-3 and C-10. The completeassignment of the protons and carbons was achieved by a combination of¹H, ¹³C, HSQC, ¹H—¹H COSY, HMBC and 1,1-ADEQUATE spectral analyses.Moreover, NOEs were observed between the methylene protons at C-1′″ andC-4′″, indicating the E-configuration of the hemiterpene moiety. Thestructure of compound 4 was identified as1″-O-[(E)-2′″-methyl-but-2′″-en-4′″-ol]-4″,6″-O-[3,5-dicarbonyl-8-hydroxy-2-(4′,5′-dihydroxy-phenyl)-1-benzofuran-2-yl]-β-D-glucopyranoseand was named salviniside I.

Compound 5 were, isolated by HPLC, obtained as colorless powders andshowed an [M-1]⁺ at m/z 473.0714 (calcd for 473.0720) in the HR-ESI-MS,suggesting the molecular formula to be C₂₂H₁₈O₁₂. The NMR spectroscopicdata of 5 (Table 5), was different from those of compound 4 mainly inthe absence of the signals for the hemiterpene moiety, which wasattached at the C-1 position of the glucose. Instead, compound 5displayed similarly two set of glycoside signals (Table 5) with twoanomeric proton at δ 4.57 (1H, d, J=7.8 Hz, β-H-1″) and δ 5.14 (1H, d,J=3.7 Hz, α-H-1″), and two protonated carbon at δ_(C-β) 98.6 and δ_(C-α)94.4 observed in the HSQC spectrum. Acid hydrolysis of compound 5liberated D-glucose identified by co-HPLC with authentic sugar (Oiso,2001; Toyota, 2002). The analysis of the COSY, HSQC spectra indicatedcompound 5 is an isomeric mixture of α and β-D-glucopyranosides(Choudhary, 2008; Narasimhulua, 2010; Ding, 1999). This conclusion wasconfirmed by production of compound 5a with β-anomeric methyl glucoseupon methylation of compound 5 with MeOH in 10% H₂SO₄. In the NMRspectrum, each signals of the aglycon part of compound 5 appearedessentially in duplicate owing to the formation of a mixture of α andβ-anomers. The complete assignment of the protons and carbons wasachieved by a combination of ¹H, ¹³C, HSQC, ¹H—¹H COSY and HMBC spectralanalyses. Thus, compound 5 was identified as4″,6″-O-[3,5-dicarbonyl-8-hydroxy-2-(4′,5′-dihydroxy-phenyl)-1-benzofuran-2-yl]-α/β-D-glucopyranoseand named salviniside II.

Compound 6 was isolated as a colorless powder. The molecular formulaC₃₀H₄₆O₂ was deduced from the [M-H]⁺ peak in the HR-ESI-MS, suggestingthe presence of eight degrees of unsaturation. The ¹³C-NMR spectrumindicated the presence of four double bonds [δ_(C) 150.9 (s), 148.8 (s),136.4 (s), 131.6 (s), 127.1 (d), 120.9 (d), 110.8 (d)] and an oxygenatedcarbon [δ_(C) 72.1 (s)]. Its ¹HNMR spectrum had signals for threetertiary methyl groups (δ_(H) 0.87, 0.89, 1.12), two isopropyl groups[δ_(H) 0.93, 0.95, 1.23, 1.24 (1H each, d, J=6.9 Hz)], an olefinicproton (δ_(H) 5.35, br. s), and two aromatic protons [δ_(H) 6.60, 6.87(1H each, s)] (Table 6).

TABLE 6 ¹³C and ¹H NMR data of compound 6 (in CDCl₃)^(a) 6 Positionδ_(C) (ppm) δ_(H) (ppm)  1 38.8 2.15 (1H, m) 1.33 (1H, m)  2 19.4 1.72(1H, m) 1.60 (1H, m)  3 41.8 1.48 (1H, m) 1.19 (1H, m)  4 33.4  5 44.91.39 (1H, m)  6 21.9 1.65 (2H, m)  7 35.3 2.93 (1H, brq, J = 7.5 Hz)  8131.6  9 148.8 10 38.0 11 110.8 6.60 (1H, s) 12 150.9 13 131.6 14 127.16.87 (1H, s) 15 27.0 3.08 (1H, sept. J = 6.9 Hz) 16 22.8 1.24 (3H, d, J= 6.9 Hz) 17 22.6 1.23 (3H, d, J = 6.9 Hz) 18 33.8 0.89 (3H, s) 19 21.70.87 (3H, s) 20 25.1 1.12 (3H, s)  1′ 136.4  2′ 120.9 5.35 (br s)  3′34.8 2.23 (1H, m) 1.95 (1H, m)  4′ 72.1  5′ 31.2 1.74 (1H, m) 1.56 (1H,m)  6′ 25.0 2.19 (2H, m)  7′ 36.6 2.92 (1H, m)  8′ 17.0 0.93 (3H, d, J =6.9 Hz)  9′ 16.9 0.95 (3H, d, J = 6.9 Hz) 10′ 46.3 2.21 (2H, m) ^(a)Theassignment was based upon COSY, HSQC, and HMBC experiments.

Those data suggested that 6 was a derivative of ferruginol (14) (Tezuka,1998) exhibiting typical signals of ferruginol: an isopropyl groupattached to a phenyl group; two para aromatic protons and a typicalH_(β)-1 proton; as well as three singlet methyl groups (Kazuko, 2006).Therefore, the other moiety of compound 6 was a cyclic monoterpenehaving a tri-substituted double bond (δ_(C) 136.4, 120.9), an isopropylgroup, and an oxygenated carbon, suggesting it to be menth-1-en-4-ol. AnHMBC experiment revealed long-range couplings from H₂-10′ at δ_(C) 46.3to C-6, C-7, C-2′ and C-6′, establishing the linkage between ferruginoland menth-1-en-4-ol. The stereochemistry of compound 6 established byNOESY experiment (Kazuko, 2006; Hsieh, 2006). Thus the structure of7-(menth-1-en-4-ol)-ferruginol was established as compound 6. Recently,abietane-type diterpenes with novel skeletons, such as diterpenesattached to sesquiterpenes (Kazuko, 2006; Hsieh, 2006; Kazuko & Kentaro,2006), dimeric diterpenes (Shigenobu, 2004), and monoterpenes (Wu, 2010)were reported from the bark and heartwood of Cryptomeria japonica (L.f.) D. Don, the fruits of C. fortunei Hooibr. ex Otto & A. Dietr., andthe bark of Calocedrus macrolepis var. formosana (Florin) W. C. Cheng &L. K. Fu. Compound 6 has a new ferruginol-menthol skeleton and is namedas salviniol (6) because of the plant source Salvinia.

Example 3

All the isolated compounds (1-50) were further tested for theircytotoxicity against human exocrine pancreatic cancer (PANC-1 andBxPC-3), non-small cell lung cancer (A549), prostate cancer (PC-3),breast cancer (MDA-MB-231), leukemia (HL-60), and normal lung cells(MRC-5). Among all tested compounds, 16 abietane diterpenes (6-17 and19-22) demonstrated only moderate or weak activities against all tumorcells (Table 7).

TABLE 7 Cytotoxic activity of 17 abietane diterpenes against varioushuman tumor cell lines (GI₅₀) (μM) MDA- Compound A549 PC-3 HL-60 PANC-1BxPC-3 MB-231 MRC-5 6 62.91 ± 9.63 54.19 ± 5.90  48.05 ± 10.86 60.44 ±16.17  87.28 ± 24.51 75.0 ± 1.37 58.41 ± 6.83 7  9.10 ± 2.47  8.39 ±0.86 13.33 ± 5.62 22.59 ± 4.18  37.28 ± 7.44 >100 24.75 ± 4.73 8 11.07 ±3.16 13.17 ± 2.03 11.45 ± 3.13 23.08 ± 9.16   25.0 ± 13.19 N/A 20.01 ±5.14 9  84.52 ± 18.73 >100 92.78 ± 9.98 >100 >100 N/A >100 10 86.50 ±9.54 N/A N/A N/A N/A N/A N/A 11 21.21 ± 6.21 N/A N/A N/A N/A N/A N/A 1235.04 ± 6.20 41.03 ± 7.90 35.26 ± 0.63 43.14 ± 8.32  45.01 ± 8.33 N/A40.19 ± 0.91 13  79.8 ± 2.18 >100  68.64 ± 13.46 87.94 ± 11.18 36.08 ±1.50 N/A >100 14 30.67 ± 5.22  56.45 ± 16.51 27.33 ± 8.95 41.13 ± 14.1121.14 ± 0.51 N/A 40.01 ± 7.74 15 75.11 ± 7.04 85.61 ± 5.30 75.19 ± 1.0278.24 ± 13.46  74.73 ± 11.09 N/A 70.49 ± 7.21 16 53.79 ± 9.65 58.12 ±3.28 66.52 ± 0.04 46.71 ± 10.98 37.50 ± 4.01 N/A 59.25 ± 7.56 17 42.22 ±3.89  43.60 ± 11.88  50.01 ± 15.23 37.53 ± 5.72  36.42 ± 8.08 N/A 55.36± 4.94 18 (−) (−) (−) (−) (−) N/A (−) 19 24.89 ± 5.94 18.97 ± 5.34 23.27± 6.66 32.15 ± 2.75  20.54 ± 2.00 N/A 20.39 ± 3.36 20 30.14 ± 3.64 27.07 ± 11.76 36.33 ± 7.14 52.55 ± 18.84  51.04 ± 10.44 N/A 45.70 ±2.32 21 47.96 ± 7.75 46.32 ± 6.60 30.03 ± 8.30 51.34 ± 10.04 41.74 ±2.92 N/A 40.19 ± 2.06 22  93.75 ± 21.39 N/A N/A N/A N/A N/A N/A Note:GI₅₀ (mean ± S.D.) refers to the concentration required to have 50%cell-growth inhibition; (−) indicates that the isolated is inactive(negative at 100 or GI₅₀ >100 μM); N/A: no data available.

14-deoxycoleon U (7) and montbretol (8) were found to be the bestactivities and high selective, especially, no toxic against normal cell.The suppressive effects of salviniol (6) and montbretol (8) on humanPANC-1 and BxPC-3 pancreatic cancer cell proliferation wereinvestigated, and both could inhibit human pancreatic cancer cellproliferation in a dose-dependent manner (FIG. 3).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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The invention claimed is:
 1. A method of treating cancer in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising an effective amount of a compound of the formula:


2. The method of claim 1, wherein the cancer is melanoma, cervicalcancer, breast cancer, ovarian cancer, prostate cancer, testicularcancer, urothelial carcinoma, bladder cancer, non-small cell lungcancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma,gastrointestinal stromal tumors, gastroesophageal carcinoma, colorectalcancer, pancreatic cancer, kidney cancer, hepatocellular cancer,malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome,multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacell neoplasms, Wilm's tumor, or hepatocellular carcinoma.
 3. The methodof claim 1 wherein said patient is further administered a distinctcancer therapy.
 4. The method of claim 1, wherein the compound isisolated from a plant in the Salviniaceae family.
 5. The method of claim4, wherein the compound is isolated from a plant in the Salvinia genus.6. The method of claim 4, wherein the compound is isolated from S.auriculata, S. biloba, S. cucullata, S. cyathiformis, S. hastate, S.herzogii, S. minima, S. molesta, S. natans, S. nymphellula, S.oblongifolia, S. radula, S. rotundifolia, or S. sprucei.
 7. The methodof claim 4, wherein the compound is isolated from a plant in the Azollagenus.
 8. The method of claim 1, wherein the pharmaceutical compositionfurther comprises an excipient.